The objective of this project is to identify and optimize methods that can be used in a clinical cell processing laboratory to isolate, culture, genetically manipulate and cryopreserve cells which may be useful in gene therapy of diseases. As a model system, this project will utilize the retroviral transduction of the cDNA for iduronate-2-sulfatase (IDS) by a well characterized vector construct, L2SN, for the treatment of Hunter syndrome (mucopolysaccharidosis type II). Studies performed on a small scale have shown that patients with Hunter syndrome will likely benefit from ex vivo lymphocyte gene therapy with the IDS gene (project III); however, methods must be developed to produce the large numbers of cells necessary for a clinical trial (AIM 1). Only methods appropriate for use in clinical cellular therapy laboratories and for patient care will be used. Products specifically designed to effectively isolate and culture large numbers of cells will be tested. Since gene therapy with lymphocytes is most advanced at this time, initial studies will test methods to optimize the expansion of lymphocytes and their transduction with the IDS gene using a L2SN retroviral vector. For the expansion of lymphocytes, closes systems utilizing bags or hollow fibers will be compared. Fore gene transduction the same bags and hollow fiber systems will be tested; however, in addition a novel bag with two chambers separated by a membrane permeable to virus will also be tested. Studies will also be performed to optimize the isolation and transduction of hematopoietic stem cells (AIM 2). Hematopoietic stem cells (HSC) will be obtained from four sources: marrow, peripheral blood, peripheral blood of people treated with G-CSF and umbilical cord blood. Because HSC from these various sources are anticipated to have different properties, different growth factor requirements, and may respond differently to retroviral transduction, stem cells from all of these sources will be studied. Several systems designed to isolate CD34+ cells will be compared including flasks, beads and columns containing CD34 antibodies and counter flow centrifugal elutriation. The transduction of HSC in the single chamber gabs, double chamber bags and hollow fiber systems will be compared. The L2SN retroviral vector and new retroviral and adenovirus associated virus (AAV) vectors developed by projects I and II will be tested. Since patients will be treated several times with transduced lymphocytes or stem cells, methods will be developed to cryopreserve enough transduced cells for several treatments (AIM 3). Although Dimethyl sulfoxide (DMSO) can be used to cryopreserve lymphocytes and HSC, DMSO is toxic. Hydroxyethyl starch, propylene glycol, and glycerol all of which are approved for use in humans will be tested as cryoprotective agents. The effects of the duration of storage on cryopreserved cells will also be investigated.